Modified lipase and use thereof

ABSTRACT

The present invention addresses a problem of providing a lipase derived from a microorganism that is specific for short-chain to medium-chain fatty acids. A modified lipase is obtained by making a substitution in the amino acid sequence of a  Candida cylindracea  derived lipase, wherein the substitution is (1) a substitution of asparagine for an amino acid corresponding to the amino acid at position 428 in the amino acid sequence set forth in SEQ ID NO: 1; or (2) a substitution of phenylalanine, methionine, or isoleucine for an amino acid corresponding to the amino acid at position 429 in the amino acid sequence set forth in SEQ ID NO: 1.

The present invention relates to a modified lipase. Specifically, thepresent invention provides, for example, a modified lipase and a methodfor producing dairy products using such a modified lipase. The presentapplication is a Division Application of U.S. Ser. No. 15/102,324 filedon Jun. 7, 2016, issued as U.S. Pat. No. 10,415,023, which is a nationalstage entry of PCT/JP2014/082415, filed on Dec. 8, 2014, which claimspriority from Japanese Patent Application No. 2013-255419, filed on Dec.10, 2013. The entire contents of these applications are incorporatedherein by reference in their entirety.

TECHNICAL FIELD Background Art

Lipases have been used for formation and enhancement of the flavor ofdairy products. Traditionally, there have been used preparations oflipases from kids, calves, or lambs. These ruminant lipases have aspecificity that short-chain fatty acids (C₄ and C₆ fatty acids) arereleased from milk fat, and are suitable for the formation of the flavorof dairy products.

However, there is a strong industrial need for an alternative toanimal-derived lipases because kosher or halal qualities are requiredfor enzyme preparations utilized for food processing. To meet the need,proposals have been made, for example, to use microbial enzymes (forexample, Patent Document 1) and recombinant enzymes (for example, PatentDocument 2). In addition, attempts have also been made to modify lipasesby genetic engineering, for application to particular purposes (forexample, Patent Documents 3 to 5).

CITATIONS LIST Patent Literatures

-   Patent Literature 1: JP 61-135541 A-   Patent Literature 2: US 2004/0001819-   Patent Literature 3: JP 2011-512809-   Patent Literature 4: JP 2003-524386-   Patent Literature 5: JP 2004-517639

Non Patent Literature

-   Non Patent Literature 1: J. Schmitt et al., Protein Engineering,    vol. 15, no. 7, pp. 595-601, 2002

SUMMARY OF INVENTION Technical Problems

Microbial lipases are more specific for long-chain fatty acids than forshort-chain fatty acids, and thus their action on milk fat will give aprofile that many of the fatty acids released from the milk fat have along chain. Long-chain fatty acids, which are responsible for soap odor,are not favorable as the favor of dairy products, particularly ofcheeses.

Under this background, the present invention addresses a problem ofproviding a lipase derived from a microorganism that is specific forshort-chain to medium-chain fatty acids, and a use of such a lipase.

Solutions to Problems

In the course of the investigation to solve the above-mentioned problem,the inventors focused on a Candida cylindracea derived lipase (a lipaseformerly referred to as a Candida rugosa derived lipase was used) andattempted its modification. After trial and error, the inventorssucceeded in finding very useful mutation sites that can lead to theachievement of the goals of the present invention, from the amino acidswhich form the substrate pocket. Variants with a given amino acidsubstitution made at each of these mutation sites hydrolyzed milk fat sothat short-chain to medium-chain fatty acids (C₄ to C₈ fatty acids) wereselectively released as in the case of an animal lipase. These variantsworked well on short-chain fatty acids (C₄ to C₆ fatty acids), and beston C₄ fatty acid. As just mentioned, the inventors succeeded, as aresult of these amino acid mutations, in bringing the substratespecificities of lipases close to that of the animal lipase. Inconnection with this, for the Candida cylindracea derived lipase, therehave been reported mutations (amino acid substitutions) considered to beeffective for its substrate specificity (Non-Patent Document 1), but itwas observed that the newly found mutations were more effective inmodifying the substrate specificity (specificity for short-chain fattyacids).

In present invention, it is likely that mutagenesis procedures similarto those as described herein can also be applied to other enzymes havinga high degree of amino acid sequence identity relative to LIP1 used inExamples, in light of common general technical knowledge that enzymeshaving a high degree of amino acid sequence identity (typicallyisozymes) have a high degree of similarity in their three-dimensionalstructure, particularly in sites involved in their activity, such asactive site and substrate pocket, and that it is highly probable that asimilar mutation in such enzymes gives rise to a similar effect.

The inventions described below are based mainly on the above-describedresults and observation.

[1] A modified lipase consisting of an amino acid sequence with asubstitution made in the amino acid sequence of a Candida cylindraceaderived lipase, wherein the substitution is:

(1) a substitution of asparagine for an amino acid corresponding to theamino acid at position 428 in the amino acid sequence set forth in SEQID NO: 1; or

(2) a substitution of phenylalanine, methionine, or isoleucine for anamino acid corresponding to the amino acid at position 429 in the aminoacid sequence set forth in SEQ ID NO: 1.

[2] The modified lipase according to [1], wherein the amino acidsequence of the Candida cylindracea derived lipase is an amino acidsequence that is 70% or more identical to the amino acid sequence of SEQID NO: 2 and wherein the substitution is the substitution represented in(1).

[3] The modified lipase according to [1], wherein the amino acidsequence of the Candida cylindracea derived lipase is an amino acidsequence that is 90% or more identical to the amino acid sequence of SEQID NO: 2 and wherein the substitution is the substitution represented in(2).

[4] The modified lipase according to [2] or [3], wherein the amino acidsequence of the Candida cylindracea derived lipase is an amino acidsequence of any one of SEQ ID NOs: 2 to 7.

[5] The modified lipase according to [1], consisting of the aminosequence set forth in any one of SEQ ID NOs: 8 to 11.

[6] A gene encoding the modified lipase according to any one of [1] to[5].

[7] The gene according to [6], comprising the base sequence set forth inany one of SEQ ID NOs: 12 to 19.

[8] A recombinant DNA comprising the gene according to [6] or [7].

[9] A microorganism carrying the recombinant DNA according to [8].

[10] The microorganism according to [9], wherein the host is Escherichiacoli, Candida cylindracea, Aspergillus oryzae, Bacillus subtilis, orPichia pastoris.

[11] An enzyme preparation comprising the modified lipase according toany one of [1] to [5].

[12] A method for improving the flavor of a food product or food rawmaterial, characterized in that the enzyme according to any one of [1]to [5] or the enzyme preparation according to [11] is allowed to act onthe food product or food raw material.

[13] A method for producing a food product, characterized in that theenzyme according to any one of [1] to [5] or the enzyme preparationaccording to [11] is allowed to act on a food raw material orintermediate product.

[14] The method according to [12] or [13], wherein the food product is adairy product.

[15] A flavor-improving agent that is allowed to act on a food productor food raw material, comprising the enzyme according to any one of [1]to [5] or the enzyme preparation according to [11].

[16] A food product or food raw material obtained by treatment with theenzyme according to any one of [1] to [5] or the enzyme preparationaccording to [11].

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the composition of released fatty acids after treatmentwith a wild-type enzyme. A Candida cylindracea derived wild-type lipaseLIP1 was allowed to act on cheese (used as a substrate) and thecomposition of the released fatty acids was analyzed.

FIG. 2 shows the composition of released fatty acids after treatmentwith modified enzymes. Various modified lipases (variants) were allowedto act on cheese (used as a substrate) and a comparison was made for thecompositions of the released fatty acids. The upper left panel showsresults when a calf sublingual gland derived lipase was used. The lowerpanels show results when modified lipases were used (variant 1: L428N;variant 2: G429F, variant 3: G429M; and variant 4: G429I). The upperright panel shows results when a modified lipase (L428F) that hadpreviously been reported was used.

FIG. 3 shows a comparison of the sequences of Candida cylindraceaderived wild-type lipases LIP1 (SEQ ID NO: 2), LIP1′ (SEQ ID NO: 3),LIP2 (SEQ ID NO: 4), LIPS (SEQ ID NO: 5), LIP4 (SEQ ID NO: 6), and LIPS(SEQ ID NO: 7).

FIG. 4 is a continuation of FIG. 3.

FIG. 5 is a continuation of FIG. 4.

DESCRIPTION OF EMBODIMENTS

For convenience of description, some of the terms used in relation tothe present invention are defined as follows.

Terminology

The term “modified lipase” refers to an enzyme obtained by modificationor mutation of a particular lipase (which is referred to as a “referencelipase” for convenience of description). The reference lipase is aCandida cylindracea derived lipase or a Candida rugosa derived lipase.The terms “Candida cylindracea derived lipase” and “Candida rugosaderived lipase” are used interchangeably.

The term “Candida cylindracea derived lipase” is a lipase that isobtained from a strain of Candida cylindracea as the source, andincludes lipases produced by Candida cylindracea, lipases produced bymutated strains of Candida cylindracea (variant strains), lipasesexpressed, for example, in other microorganism, using the geneticinformation of such enzymes, or the like. Similarly, the term “Candidarugosa derived lipase” is a lipase that is obtained from a strain ofCandida rugosa as the source, and includes lipases produced by Candidarugosa, lipases produced by mutated strains of Candida rugosa (variantstrains), lipases expressed, for example, in other microorganism, usingthe genetic information of such enzymes, or the like.

In the present invention, an “amino acid substitution” is carried out asmodification or mutation. Therefore, some amino acid residues are foundto be different when a modified lipase and the reference lipase thereforare compared. In the specification, a modified lipase is also referredto as a modified enzyme or as a variant.

In the specification, amino acids are designated according to the commonpractice, as their single letters as described below:

methionine: M; serine: S; alanine: A; threonine: T; valine: V; tyrosine:Y; leucine: L; asparagine: N; isoleucine: I; glutamine: Q; proline: P;aspartic acid: D; phenylalanine: F; glutamic acid: E; tryptophan: W;lysine: K; cysteine: C; arginine: R; glycine: G; and histidine: H.

In the specification, the positions of amino acids in an amino acidsequence are specified by assigning the numbers from the N-terminustoward the C-terminus of the amino acid sequence, wherein according tocustomary practice, the methionine corresponding to the translationinitiation site is assigned to 1, i.e., the first amino acid. Therefore,in the case of the sequence of a mature protein in which the signalpeptide has been removed, the amino acid numbers are decreased by thenumber of the amino acids of the signal peptide.

In the specification, an amino acid residue at a mutation site (an aminoacid residue to be substituted with another amino acid) is expressed ina combination of the above-described single letter representing the kindof the amino acid residue and the figure representing the position ofthe amino acid residue. For example, if proline at position 428 is amutation site, then the amino acid is designated as “G428.”

1. Modified Lipases

A first aspect of the present invention is directed to a modified lipase(modified enzyme). The modified enzyme of the present invention has anamino acid sequence with a substitution made in the amino acid sequenceof a Candida cylindracea derived lipase, wherein the substitution is:

(1) a substitution of asparagine for an amino acid corresponding to theamino acid at position 428 in the amino acid sequence set forth in SEQID NO: 1; or

(2) a substitution of phenylalanine, methionine, or isoleucine for anamino acid corresponding to the amino acid at position 429 in the aminoacid sequence set forth in SEQ ID NO: 1.

The sequence of SEQ ID NO: 1 is the amino acid sequence of a Candidacylindracea derived lipase LIP 1, which comprises the signal peptide. Inthe substitution represented in (1), an amino acid corresponding to theamino acid at position 428 in this amino acid sequence is a target to besubstituted with a given amino acid and is substituted with asparagine,resulting in an alteration of the substrate specificity of the lipase.In the substitution represented in (2), an amino acid corresponding tothe amino acid at position 429 in the sequence of SEQ ID NO: 1 is atarget to be substituted with a given amino acid and is substituted withphenylalanine, methionine, or isoleucine, resulting in an alteration ofthe substrate specificity of the lipase. Lipases after these amino acidsubstitutions, i.e., modified enzymes have an increased specificity forshort-chain to medium-chain fatty acids (C₄ to C₈ fatty acids), and whenallowed to act on milk fat, typically give a composition of the releasedfatty acids that is similar to that of a calf sublingual gland derivedlipase. Preferably, these modified enzymes work well on short-chainfatty acids (C₄ to C₆ fatty acids), and best on C₄ fatty acid.

Herein, the term “corresponding” when used for an amino acid residue inthe present specification means contributing equally to exhibition offunctions among proteins (enzymes) being compared. For example, when anamino acid sequence for comparison to the base amino acid sequence (thatis, the amino acid sequence set forth in SEQ ID NO: 1) is aligned whileconsidering partial homology of the primary structure (that is, an aminoacid sequence) so that the most appropriate comparison can be achieved(in this event, the alignment may be optimized by introducing gaps ifnecessary), an amino acid located at a position corresponding to aspecific amino acid in the base amino acid sequence can be specified asa “corresponding amino acid”. The “corresponding amino acid” can also bespecified by comparison between conformations (three-dimensionalstructures) in place of or in addition to the comparison between primarystructures. Utilization of conformational information can give highlycredible comparison results. In this case, a technique of performing analignment with comparing atomic coordinates of conformations of aplurality of enzymes can be adopted.

Conformational information of an enzyme to be mutated is available from,for example, the Protein Data Bank Japan.

One example of a method for determination of a protein conformation bythe X-ray crystal structure analysis will be shown below.

(1) A protein is crystallized. Crystallization is essential to determinea conformation, and in addition, crystallization is industrially usefulas a purification method of a protein at high purity and a stablepreservation method of a protein at high density. In this case, aprotein to which a substrate as a ligand or its analogous compound isbound may be preferably used for crystallization.

(2) The prepared crystal is irradiated with X ray to collect diffractiondata. There are many cases that a protein crystal is damaged due to Xray irradiation and the diffraction ability is deteriorated. In suchcases, a low-temperature measurement technique of rapidly cooling thecrystal to about −173° C. and collecting diffraction data in the statehas been recently prevailed. In addition, ultimately, synchrotron orbitradiation having high luminance is utilized to collect high resolutiondata that is used for structural determination.

(3) In addition to the diffraction data, phase information is necessaryin order to perform the crystal structure analysis. When a crystalstructure of an analogous protein to a desired protein is unknown, it isimpossible to determine the structure in a molecular substitutionmethod, and a phase problem has to be solved by a heavy-atom isomorphousreplacement method. The heavy-atom isomorphous replacement method is amethod in which a metallic atom having a high atomic number such asmercury or platinum is introduced into a crystal and contribution of alarge X ray scattering ability of such a metallic atom to X raydiffraction data is utilized to collect phase information. Thedetermined phase is possibly improved by smoothing an electron densityof a solvent region in the crystal. Since a water molecule in thesolvent region has large fluctuation, the electron density is hardlyobserved, and thus adjusting the electron density in this region toclose to 0 makes it possible to approach the real electron density,which results in improving a phase. When plural molecules are containedin an asymmetrical unit, equation of electron densities of thesemolecules makes it possible to more significantly improve a phase. Amodel of a protein is fit to an electron density map calculated usingthe phase improved as described above. This process is performed oncomputer graphics using a program such as QUANTA made by MSI Co. (USA).After the process, structural precision is performed using a programsuch as X-PLOR made by MSI Co. to complete the structure analysis. Whena crystal structure of an analogous protein to a desired protein isknown, it can be determined in a molecular substitution method using theatomic coordinate of the known protein. Molecular substitution andstructure refinement can be performed using a program such as CNS_SOLVEver.11.

As Candida cylindracea derived lipases, there are known five enzymes(LIP1, LIP2, LIPS, LIP4, and LIP5). In addition, the applicant has foundan enzyme (referred to as LIP1′) that exhibits a high homology to LIP1,from lipase-producing mutant strains. For these six enzymes, the aminoacid sequences without the signal peptide, i.e., the amino acidsequences of the mature enzymes are set forth in SEQ ID NOs: 2 (forLIP1), 3 (for LIP1′), 4 (for LIP2), 5 (for LIP3), 6 (for LIP4), and 7(for LIP5). Typically, one of these enzymes will be used as a referencelipase (which is subjected to amino acids substitutions, resulting inthe generation of modified enzymes). Therefore, specific examples of theamino acid sequence of a reference lipase are the amino acid sequencesof SEQ ID NOs: 2 to 7. The identity to the amino acid sequence ofCandida cylindracea derived lipase LIP1 (SEQ ID NO: 2) is 99% for theamino acid sequence of SEQ ID NO: 3, 79% for the amino acid sequence ofSEQ ID NO: 4, 88% for the amino acid sequence of SEQ ID NO: 5, 81% forthe amino acid sequence of SEQ ID NO: 6, and 82% for the amino acidsequence of SEQ ID NO: 7 (FIGS. 3 to 5).

For the substitution represented in (1) (a substitution for an aminoacid corresponding to the amino acid at position 428 in the amino acidsequence of SEQ ID NO: 1), an enzyme consisting of an amino acidsequence 70% or more identical to the amino acid sequence of SEQ ID NO:2 may be used as a reference lipase. For example, any of LIP1, LIP1′,LIP2, LIP3, LIP4, and LIP5 can be the reference lipase. As a referencelipase, use is preferably made of an enzyme that has an amino acidsequence having 80% or more identity to the amino acid sequence of SEQID NO: 2 (with the proviso that the enzyme exhibits lipase activity),more preferably an enzyme that has an amino acid sequence having 90% ormore identity to the amino acid sequence of SEQ ID NO: 2 (with theproviso that the enzyme exhibits lipase activity), even more preferablyan enzyme that has an amino acid sequence having 95% or more identity tothe amino acid sequence of SEQ ID NO: 2 (with the proviso that theenzyme exhibits lipase activity), and most preferably an enzyme that hasan amino acid sequence having 99% or more identity to the amino acidsequence of SEQ ID NO: 2 (with the proviso that the enzyme exhibitslipase activity).

In LIP1 having the amino acid sequence of SEQ ID NO: 2, the amino acidcorresponding to the amino acid at position 428 in the amino acidsequence of SEQ ID NO: 1 is leucine (L) at position 413. Therefore, whenLIP1 having the amino acid sequence of SEQ ID NO: 2 is used as areference lipase, this amino acid is a target to be substituted with agiven amino acid. On the other hand, when LIP1′ having the amino acidsequence of SEQ ID NO: 3 is used as a reference lipase, the amino acidto be substituted with a given amino acid is leucine (L) that is anamino acid located at position 413 in SEQ ID NO: 3. When LIP2 having theamino acid sequence of SEQ ID NO: 4 is used as a reference lipase, theamino acid to be substituted with a given amino acid is leucine (L) thatis an amino acid located at position 413 in the amino acid sequence ofSEQ ID NO: 4. When LIP3 having the amino acid sequence of SEQ ID NO: 5is used as a reference lipase, the amino acid to be substituted with agiven amino acid is leucine (L) that is an amino acid located atposition 413 in SEQ ID NO: 5. When LIP4 having the amino acid sequenceof SEQ ID NO: 6 is used as a reference lipase, the amino acid to besubstituted with a given amino acid is leucine (L) that is an amino acidlocated at position 413 in the amino acid sequence of SEQ ID NO: 6. WhenLIPS having the amino acid sequence of SEQ ID NO: 7 is used as areference lipase, the amino acid to be substituted with a given aminoacid is leucine (L) that is an amino acid located at position 413 in theamino acid sequence of SEQ ID NO: 7.

In the meanwhile, for the substitution represented in (2) (asubstitution for an amino acid corresponding to the amino acid atposition 429 in the amino acid sequence of SEQ ID NO: 1), an enzymeconsisting of an amino acid sequence 90% or more identical to the aminoacid sequence of SEQ ID NO: 2 may be used as a reference lipase. Forexample, any of LIP1 and LIP1′ can be the reference lipase. As areference lipase, use is preferably made of an enzyme that has an aminoacid sequence having 95% or more identity to the amino acid sequence ofSEQ ID NO: 2 (with the proviso that the enzyme exhibits lipaseactivity), more preferably an enzyme that has an amino acid sequencehaving 98% or more identity to the amino acid sequence of SEQ ID NO: 2(with the proviso that the enzyme exhibits lipase activity), and mostpreferably an enzyme that has an amino acid sequence having 99% or moreidentity to the amino acid sequence of SEQ ID NO: 2 (with the provisothat the enzyme exhibits lipase activity).

In LIP1 having the amino acid sequence of SEQ ID NO: 2, the amino acidcorresponding to the amino acid at position 429 in the amino acidsequence of SEQ ID NO: 1 is glycine (G) at position 414. Therefore, whenLIP1 having the amino acid sequence of SEQ ID NO: 2 is used as areference lipase, this amino acid is a target to be substituted with agiven amino acid. On the other hand, when LIP 1′ having the amino acidsequence of SEQ ID NO: 3 is used as a reference lipase, the amino acidto be substituted with a given amino acid is glycine (G) that is anamino acid located at position 414 in SEQ ID NO: 3.

Here, specific examples of the amino acid sequences of modified enzymesare represented in SEQ ID NOs: 8 to 11. A modified enzyme having theamino acid sequence of SEQ ID NO: 8 (variant 1) is obtained by making asubstitution of asparagine for an amino acid at position 413 on LIP1having the amino acid sequence of SEQ ID NO: 2 (that is, a substitutionrepresented in (1)); a modified enzyme having the amino acid sequence ofSEQ ID NO: 9 (variant 2) is obtained by making a substitution ofphenylalanine for an amino acid at position 414 on LIP1 having the aminoacid sequence of SEQ ID NO: 2 (that is, one of the substitutionrepresented in (2)); a modified enzyme having the amino acid sequence ofSEQ ID NO: 10 (variant 3) is obtained by making a substitution ofmethionine for an amino acid at position 414 on LIP1 having the aminoacid sequence of SEQ ID NO: 2 (that is, one of the substitutionrepresented in (2)); and a modified enzyme having the amino acidsequence of SEQ ID NO: 11 (variant 4) is obtained by making asubstitution of isoleucine for an amino acid at position 414 on LIP1having the amino acid sequence of SEQ ID NO: 2 (that is, one of thesubstitution represented in (2)).

In cases where a portion of the amino acid sequence of a given proteinhas been subjected to mutagenesis, a mutated version of the protein mayhave a function equivalent to the original unmutated protein. That is,it is sometimes observed that a mutation in a given amino acid sequencedoes not lead to substantial effects on the function of the protein,which is maintained between before and after introducing the mutation.Taking this common general technical knowledge into account, it can beconsidered that when compared to an above-described modified enzyme (anyof variants 1 to 4), a modified enzyme which has a slight difference inthe amino acid sequence (wherein the difference in the amino acidsequence is located at a position(s) other than the position at whichthe above-described amino acid substitution has been performed), butwhich nevertheless does not have substantial differences in propertiesis an enzyme that is substantially the same as the above-describedmodified enzyme. The “slight difference in the amino acid sequence” inthis context typically refers to the occurrence of a mutation(s)(change(s)) in the amino acid sequence resulting from deletion orsubstitution of one to several amino acids (for example, up to three,five, seven, or ten amino acids) contained in the amino acid sequence,or addition or insertion of one to several amino acids (for example, upto three, five, seven, or ten amino acids), or combinations thereof. Theidentity (%) between the amino acid sequences of “an enzyme that issubstantially the same” and an above-described modified enzyme that isused as the reference is, for example, 90% or more, preferably 95% ormore, more preferably 98% or more, most preferably 99% or more.Differences in the amino acid sequence may occur at more than oneposition. A “slight difference in the amino acid sequence” preferablyresults from conservative amino acid substitution.

2. Nucleic acid coding for modified lipase, etc.)

The second aspect of the present invention provides a nucleic acidrelating to the modified enzyme of the invention. That is, provided area gene coding for the modified enzyme, a nucleic acid that can be usedas a probe for identifying a nucleic acid coding for the modifiedenzyme, and a nucleic acid that can be used as a primer for amplifyingor mutating a nucleic acid coding for the modified enzyme.

The gene coding for a modified enzyme is typically used in preparationof the modified enzyme. According to a genetic engineering procedureusing the gene coding for a modified enzyme, a modified enzyme in a morehomogeneous state can be obtained. Further, the method can be apreferable method also in the case of preparing a large amount of amodified enzyme. Note that uses of the gene coding for a modified enzymeare not limited to preparation of a modified enzyme. For example, thenucleic acid can also be used as a tool for an experiment intended forclarification of action mechanisms of a modified enzyme or a tool fordesigning or preparing a further modified form of an enzyme.

The “gene coding for a modified enzyme” herein refers to a nucleic acidcapable of obtaining the modified enzyme when it is expressed, andincludes, as a matter of course of a nucleic acid having a base sequencecorresponding to the amino acid sequence of the modified enzyme, also anucleic acid obtained by adding a sequence that does not code for anamino acid sequence to such a nucleic acid. Degeneracy of a codon isalso considered.

Examples of the (base) sequence of the gene encoding a modified enzymeare represented in SEQ ID NOs: 12 to 15. These sequences encode variantsdescribed in the Examples section which follows, as indicated below.

SEQ ID NO: 12: variant 1 (L428N)

SEQ ID NO: 13: variant 2 (G429F)

SEQ ID NO: 14: variant 3 (G429M)

SEQ ID NO: 15: variant 4 (G429I)

In Candida cylindracea, the CTG codon encodes serine. If a gene isrecombinantly expressed using other yeasts and the like as a host, thenit is necessary that depending on the host to be used, the CTG codon ischanged to another codon encoding serine (TCT, TCC, TCA, ATG, or AGC).The present invention also provides, as the sequence of a gene for usein heterologous expression, a sequence in which a codon substitution ofthis type is made for the sequence of any one of SEQ ID NOs: 12 to 15.Examples of sequences with a codon substitution are as follows.

SEQ ID NO: 16, which is a sequence with a codon substitution in thesequence of SEQ ID NO: 12;

SEQ ID NO: 17, which is a sequence with a codon substitution in thesequence of SEQ ID NO: 13;

SEQ ID NO: 18, which is a sequence with a codon substitution in thesequence of SEQ ID NO: 14; and

SEQ ID NO: 19, which is a sequence with a codon substitution in thesequence of SEQ ID NO: 15.

When a gene according to the present invention is to be expressed in ahost, the gene will usually be inserted into the host in the form of agene construct in which the above-described sequence has a signalpeptide-coding sequence (a signal sequence) added thereto at the 5′ end.The signal sequence of wild-type LIP1 is represented in SEQ ID NO: 21.The amino acid sequence encoded by this signal sequence (that is, thesignal peptide) is represented in SEQ ID NO: 22. The signal sequence maybe selected depending on the host to be used. Any signal sequence thatcan express a variant of interest can be used in the present invention.Examples of the signal sequence that can be used in the presentinvention can be illustrated by the following: a sequence encoding thesignal peptide of the α-factor (Protein Engineering, 1996, vol. 9, p.1055-1061), a sequence encoding the signal peptide of the α-factorreceptor, a sequence encoding the signal peptide of the SUC2 protein, asequence encoding the signal peptide of the PHOS protein, a sequenceencoding the signal peptide of the BGL2 protein, a sequence encoding thesignal peptide of the AGA2 protein, a sequence encoding the signalpeptide of TorA (trimethylamine N-oxidoreductase), a sequence encodingthe signal peptide of Bacillus subtilis derived PhoD (phosphoesterase),a sequence encoding the signal peptide of Bacillus subtilis derived LipA(lipase), a sequence encoding the signal peptide of Aspergillus oryzaederived Taka-amylase (JP 2009-60804 A), a sequence encoding the signalpeptide of Bacillus amyloliquefaciens derived α-amylase (Eur. J.Biochem. 155, 577-581 (1986)), a sequence encoding the signal peptide ofBacillus subtilis derived neutral protease (APPLIED AND ENVIRONMENTALMICROBIOLOGY, April 1995, p. 1610-1613, Vol. 61, No. 4), and a sequenceencoding the signal peptide of Bacillus derived cellulase (JP2007-130012 A).

The nucleic acid of the present invention can be prepared in an isolatedstate by use of a standard genetic engineering technique, molecularbiological technique, biochemical technique, and the like in referenceto the present specification or the sequence information disclosed inthe appended sequence listing.

Another embodiment of the present invention provides a nucleic aciddifferent in a base sequence in a part (hereinafter also referred to asa “homologous nucleic acid”, and a base sequence defining a homologousnucleic acid is also referred to as a “homologous base sequence”) ascompared to the base sequence of the gene coding for the modified enzymeof the invention, although functions of a protein coded by the nucleicacid are equal. An example of the homologous nucleic acid includes a DNAcomposed of a base sequence containing substitution, deletion,insertion, addition or inversion of 1 to several bases on the basis ofthe base sequence of the nucleic acid coding for the modified enzyme ofthe present invention and coding for a protein having activity which ischaracteristic to the modified enzyme (i.e. lipase activity).Substitution or deletion of bases may occur in a plurality of sites. The“plurality” herein depends on positions or kinds of amino acid residuesin a conformation of a protein coded by the nucleic acid but means, forexample, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2to 10 bases.

Such a homologous nucleic acid as described above can be obtained by,for example, a restriction enzyme treatment, a treatment withexonuclease, DNA ligase, etc., and introduction of mutation by a sitedirected mutation introduction method (Molecular Cloning, Third Edition,Chapter 13, Cold Spring Harbor Laboratory Press, New York), and randommutation introduction method (Molecular Cloning, Third Edition, Chapter13, Cold Spring Harbor Laboratory Press, New York). The homologousnucleic acid can be obtained also in other methods such as exposure toultraviolet radiation.

Another embodiment of the present invention relates to a nucleic acidhaving a base sequence complementary to the base sequence of the genecoding for the modified enzyme of the invention. Another embodiment ofthe present invention provides a nucleic acid having a base sequencewith an identity of at least about 60%, 70%, 80%, 90%, 95%, 99%, or99.9% to the base sequence of the gene coding for the modified enzyme ofthe invention or a base sequence complementary to the base sequence.

Another embodiment of the present invention relates to a nucleic acidhaving a base sequence hybridizing to a base sequence complementary tothe base sequence of the gene coding for the modified enzyme of theinvention or its homologous base sequence under stringent conditions.The “stringent conditions” herein refer to conditions wherein aso-called specific hybrid is formed and a nonspecific hybrid is notformed. Such stringent conditions are known by a person skilled in theart and can be set in reference to, for example, Molecular Cloning(Third Edition, Cold Spring Harbor Laboratory Press, New York) andCurrent protocols in molecular biology (edited by Frederick M. Ausubelet al., 1987). Examples of the stringent conditions include conditionsof using a hybridization liquid (50% formamide, 10× SSC (0.15 M NaCl, 15mM sodium citrate, pH 7.0), a 5× Denhardt solution, 1% SDS, 10% dextransulfate, 10 μg/ml of modified salmon sperm DNA, and a 50 mM phosphatebuffer (pH7.5)) and incubating at about 42° C. to about 50° C.,thereafter washing with 0.1× SSC and 0.1% SDS at about 65° C. to about70° C. Examples of more preferable stringent conditions includeconditions of using 50% formamide, 5× SSC (0.15 M NaCl, 15 mM sodiumcitrate, pH 7.0), a 1× Denhardt solution, 1% SDS, 10% dextran sulfate,10 μg/ml of modified salmon sperm DNA, and a 50 mM phosphate buffer (pH7.5) as a hybridization liquid.

Another embodiment of the present invention provides a nucleic acid(nucleic acid fragment) having a part of the base sequence of the genecoding for the modified enzyme of the invention or a base sequencecomplementary to the base sequence. Such a nucleic acid fragment can beused in detection, identification, and/or amplification of a nucleicacid having the base sequence of the gene coding for the modified enzymeof the present invention. For example, the nucleic acid fragment isdesigned so as to at least contain a part being hybridized to asequential nucleotide moiety (for example, about 10 to about 100 baseslength, preferably about 20 to about 100 bases length, more preferablyabout 30 to about 100 bases length) in the base sequence of the genecoding for the modified enzyme of the invention. When used as a probe,the nucleic acid fragment can be labeled. Examples such as fluorescentsubstances, enzymes, and radioactive isotopes can be used for thelabeling.

Another aspect of the present invention relates to a recombinant DNAcontaining the gene of the present invention (the gene coding for amodified enzyme). The recombinant DNA of the invention is provided in,for example, a form of a vector. The term “vector” in the presentspecification refers to a nucleic acid molecule that can transfer anucleic acid inserted in the vector to a target such as a cell.

A suitable vector is selected according to its intended use (cloning,expression of a protein) and in consideration of a kind of a host cell.Examples include a M13 phage or an altered form thereof, a λ phage or analtered form thereof, and pBR322 or an altered form thereof (e.g.,pB325, pAT153, pUC8), etc. as a vector having Escherichia coli as ahost, pYepSecl, pMFa, and pYES2 as a vector having a yeast as a host,pAc, pVL, etc. as a vector having an insect cell as a host, and pCDM8,pMT2PC, etc. as a vector having a mammal cell as a host.

The vector of the present invention is preferably an expression vector.The “expression vector” refers to a vector capable of introducing anucleic acid inserted in the expression vector into a target cell (hostcell) and expressing it in the cell. The expression vector generallycontains a promoter sequence necessary for expression of a nucleic acidinserted, an enhancer sequence for promoting expression, and the like.An expression vector containing a selective marker can also be used.When such an expression vector is used, presence or absence (and itsdegree) of introduction of the expression vector can be confirmed usinga selective marker.

Insertion of the nucleic acid of the present invention into the vector,insertion of a selective marker gene (if necessary), insertion of apromoter (if necessary), and the like can be performed in a standardrecombinant DNA technique (for example, a known method of using arestriction enzyme and a DNA ligase, which can be referred in MolecularCloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, NewYork).

As host cells, there can be employed, for example, microbial cells ofkoji mold (for example, Aspergillus oryzae), bacilli (for example,Bacillus subtilis), Escherichia coli, and Saccharomyces cerevisiae, interms of easy handling; however, any host cell in which a recombinantDNA can be replicated and a gene encoding a modified enzyme can beexpressed can be utilized. Preferably, Escherichia coli andSaccharomyces cerevisiae can be employed as a host organism. Candidayeasts such as Candida cylindracea can also be used as a host organism.In addition, Pichia yeasts such as Pichia pastoris can also be used as ahost organism. Strains of Escherichia coli can be Escherichia colistrain BL21(DE3)pLysS in cases of using a T7-based promoter, andEscherichia coli strain JM109 in other cases. Strains of Saccharomycescerevisiae can be Saccharomyces cerevisiae strain SHY2, AH22, or INVSc1(Invitrogen).

Another aspect of the present invention relates to a microorganismhaving the recombinant DNA of the invention (that is, a transformant).The microorganism of the invention can be obtained by transfection ortransformation using the vector of the invention described above. Thetransfection or transformation can be performed in, for example, thecalcium chloride method (J. Mol. Biol., 53, 159 (1970)), the Hanahanmethod (J. Mol. Biol., 166, 557 (1983)), the SEM method (Gene, 96, 23(1990)), a method by Chung, et al. (Proc. Natl. Acad. Sci. U.S.A. 86,2172 (1989)), the calcium phosphate coprecipitation method,electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81,7161-7165 (1984)), and lipofectin (Feigner, P. L. et al., Proc. Natl.Acad. Sci. U.S.A. 84, 7413-7417 (1984)). The microorganism of thepresent invention can be used for producing the modified enzyme of theinvention.

3. Enzyme Preparetion Containing Modified Lipase

The modified enzyme of the present invention is provided, for example,in the form of an enzyme preparetion. The enzyme preparetion may containan excipient, a buffer agent, a suspending agent, a stabilizer, apreservative, an antiseptic, saline and the like besides the activeingredient (the modified enzyme of the present invention). As theexcipient, starch, dextrin, maltose, trehalose, lactose, D-glucose,sorbitol, D-mannitol, white soft sugar, glycerol and the like can beused. As the buffer agent, phosphates, citrates, acetates and the likecan be used. As the stabilizer, propylene glycol, ascorbic acid and thelike can be used. As the preservative, phenol, benzalkonium chloride,benzyl alcohol, chlorobutanol, methylparaben and the like can be used.As the antiseptic, ethanol, benzalkonium chloride, paraoxybenzoic acid,chlorobutanol and the like can be used.

4. Uses of Modified Lipases

A further aspect of the present invention is directed to uses ofmodified enzymes and enzyme preparations. A modified enzyme according tothe present invention has a substrate specificity similar to that of ananimal lipase, that is, a selectivity for short-chain to medium-chainfatty acids. Taking advantage of this property, the present inventionutilizes such a modified enzyme or a preparation thereof for flavorimprovement of food products or food raw materials. “Flavor improvement”refers to providing a given product or raw material with a morefavorable flavor than its original flavor (that is, the flavor of thegiven product or raw material to which the present invention is notapplied) by increasing or adding its particular flavor component(s).Typically, flavor improvement results in the enhancement of a flavorcharacteristic of a given food product or food raw material. The flavormay be improved by masking an unfavorable flavor component(s).

Food products or food raw materials to which the present invention canbe applied can be illustrated by the following: dairy products,margarine-based products (margarines, fat spreads), shortenings, icecream-based products (ice creams, gelati, frozen yogurts, sundaes,smoothies, soft creams, etc.), ices, mousses, Bavarian creams, snacks,dressings, soups, various vegetable oils (soybean oil, rapeseed oil,corn oil, palm oil, palm kernel oil, coconut oil, sunflower oil,cottonseed oil, etc.).

For example, by allowing a modified enzyme or enzyme preparation of thepresent invention to act on a food product or food raw material, itsflavor can be improved. On the other hand, if a modified enzyme orenzyme preparation of the present invention is added to or mixed to araw material or intermediate product in a step for producing the foodproduct, then a food product with an improved flavor can be produced.Alternatively, the flavor of a food product or food raw material may beimproved, for example, by addition or mixing of a composition that isobtained using a modified enzyme or enzyme preparation of the presentinvention.

A modified enzyme or enzyme preparation of the present invention issuitable particularly for the production of dairy products. The flavorof dairy products, particularly the cheese flavor, can be increased orimproved by applying to them a modified enzyme or enzyme preparation ofthe present invention.

Examples of dairy products to which a modified enzyme or enzymepreparation of the present invention can be applied can include varioustypes of cheese (Cheshire cheese, Cheddar cheese, Edam cheese, Goudacheese, Emmental cheese, Parmesan cheese, Pecorino cheese, etc.),processed cheese (process cheese), EMC (Enzyme modified cheese), cheesefoods (which are produced by processing one or more kinds of natural orprocess cheese and have a cheese weight of 51% or higher in theproduct), butters, yogurts, creams, spreads, modified milk powders, andseasonings (to be used, for example, for snacks, dressings, and soups).Milks that are used as the main raw material for dairy products are onesfrom cows, sheep, goats, and others.

A modified enzyme or enzyme preparation of the present invention isadded, for example, to a raw material or intermediate product during thecourse of producing the food product. This allows the enzyme to act onthe milk fat present in the raw material or intermediate product,thereby leading to the release of fatty acids. The modified enzyme orenzyme preparation of the present invention can be added at variousstages in the course of producing the dairy product. Amounts(concentrations) of enzyme to be used, temperature conditions, reactiontime, and others may be determined through preliminary experiments.

EXAMPLES

A. Generation of New Lipases

The inventors carried out the investigation described below, with aimingat the generation of new lipases.

1. Objectives and Investigation Strategy

The inventors made investigations, paying attention to:

(1) Aiming at acquiring a microbial lipase that provides a similarcomposition of released fatty acids to that provided by a calfsublingual gland derived lipase when the microbial lipase is allowed toact on cheese. In particular, attempts were made to change the fattyacid specificity from long-chain to short-chain fatty acids.

(2) Making the substrate pocket of an enzyme protein small, thereby tochange the substrate specificity.

(3) Replacing an amino acid in the substrate pocket with a more bulkyamino acid, thereby to making the substrate pocket smaller.

2. Methods

(1) Selection of Mutation Sites

Amino acids that interact with substrates were selected based on thesequence of Candida cylindracea derived lipase LIP1 (its amino acidsequence including the signal peptide is represented in SEQ ID NO: 1,and the sequence of the gene encoding the amino acid sequence in SEQ IDNO: 20, and the amino acid sequence of the mature lipase without thesignal peptide in SEQ ID NO: 2) and on the three dimensional structuresdeposited in public databases. Specifically, proline at position 261(P261), leucine at position 319 (L319), and leucine at position 428(L428) were selected. These amino acid residues correspond to P246,L304, and L413, respectively, in the literature by Schmitt et al. (NonPatent Document 1: J. Schmitt et al., Protein Engineering, vol. 15, no.7, pp. 595-601, 2002).

At the same time, mutation sites were searched by computer analysis withtaking note of the increase of the hydrophobicity in the pocket toimprove the ability to synthesize esters. Serine at position 380 (S380)and glycine at position 429 (G429), which are neutral amino acids, wereselected.

(2) Acquirement of DNA Sequences Encoding Mutated Amino Acid Sequences

A Pichia pastoris host expression system (Invitrogen, Pichia ExpressionKit) was used. As a plasmid, pPIC3.5k was used. The gene for Candidacylindracea derived LIP1 that was used as a template was anLIP1-encoding sequence codon-optimized for Saccharomyces cerevisiae.Mutations were introduced by Inverse PCR method (TOYOBO,KOD-Plus-Mutagenesis Kit), thereby preparing genes encoding variousvariants with an amino acid substitution occurring at selected sites formutation. A plasmid carrying a mutated LIP1 gene was transformed into E.coli strain DH5a. Subsequently, the plasmid carrying the mutated LIP1gene was extracted from transformed E. coli cells.

(3) Acquirement of Transformants Expressing Mutated Amino Acid Sequences

The plasmid carrying the mutated LIP1 gene was transformed into Pichiapastoris strain GS115 (Invitrogen, Pichia Expression Kit). A resultingPichia pastoris transformant was cultured and the enzyme (variantlipase) was collected form the cultured supernatant.

(4) Decomposition of Milk Fat using Variant Lipases

As a substrate, natural cheese (young Cheddar cheese) was used which wassuspended and dispersed in phosphate buffer (pH 6.8) at a weight ratioof 1:1. The reaction was carried out under conditions at 50° C. for 16hours. The amount of each variant lipase to be added was 0.1 mg proteinper 1 g of cheese. After the reaction was completed, the released fattyacids in the cheese were extracted with diethyl ether and subjected togas chromatography.

From the results of evaluation on more than ten variant lipases, it wasfound that several of the variant lipases gave a composition of thereleased fatty acids that, unlike that in the case of a wild-type lipase(FIG. 1), was similar to that in the case of a calf sublingual glandderived lipase (variant 1: L428N, variant 2: G429F, variant 3: G429M,and variant 4: G429I; FIG. 2). Accordingly, the inventors have succeededin obtaining variant lipases that selectively release short-chain tomedium-chain fatty acids (C₄ to C₈ fatty acids). When allowed to act onmilk fat, these variant lipases work well on short-chain fatty acids (C₄to C₆ fatty acids), and best on C₄ fatty acid. Among these variantlipases, variant 3 is remarkable in that it is more specific forshort-chain fatty acids than the calf sublingual gland derived lipase.For comparison, the result for the variant L428F reported in theabove-mentioned literature (which is referred to therein as L413F) isshown (FIG. 2, upper right panel). Variant L428F releases long-chainfatty acids in relatively large amounts. The amino acid sequences ofthese variants and the sequences of the genes encoding them (whereincodons characteristic of Candida yeasts are used so as to correspond towild type lipase) are as follows:

<Variant 1>

Amino acid sequence: SEQ ID NO: 8

Gene sequence: SEQ ID NO: 12

<Variant 2>

Amino acid sequence: SEQ ID NO: 9

Gene sequence: SEQ ID NO: 13

<Variant 3>

Amino acid sequence: SEQ ID NO: 10

Gene sequence: SEQ ID NO: 14

<Variant 4>

Amino acid sequence: SEQ ID NO: 11

Gene sequence: SEQ ID NO: 15

B. Expression of a Variant Lipase in Various Hosts

(1) Expression of a Variant Lipase in Escherichia coli

A gene for a variant lipase (G429M) was inserted into a plasmid pET20b.The variant lipase was expressed using Escherichia coli Origami B (DE3)as a host. A resulting transformant was cultured under conditions at 15°C. for 40 hours to obtain bacterial cells. The bacterial cells weredisrupted with a Bead Shocker, and lipase activity of the resultantextract was measured. For measuring the lipase activity for short-chainfatty acids, a Lipase Kit S (DS Biopharma Medical) was used. Formeasuring the lipase activity for long-chain fatty acids, afat-digesting capacity LMAP method was used. The results revealed thatthe lipase activity of the cell extract was 1.85 u/mL when the LipaseKit S was used and 0 u/mL when the LMAP method was used.

A gene for a variant lipase (G429M) was inserted into a plasmidpCold-TF. The variant lipase was expressed using Escherichia coliOrigami B (DE3) as a host. A resulting transformant was cultured in LBmedium under conditions at 15° C. for 40 hours to obtain bacterialcells. The bacterial cells were disrupted with a Bead Shocker, andlipase activity of the resultant extract was measured. The resultsrevealed that the lipase activity of the cell extract was 3.95 u/mL whenthe Lipase Kit S was used and 0 u/mL when the LMAP method was used.

As mentioned above, a variant lipase (G429M) specific for short-chainfatty acids was able to be expressed.

(2) Expression of a Variant Lipase in a Yeast (Candida cylindracea)Strain

A variant lipase (G429M) was expressed using as a host a strain ofCandida cylindracea that had been made auxotrophic by mutagenesis. Aresulting transformant was cultured under conditions at 25° C. for 48hours, and lipase activity of the cultured supernatant was measured. Formeasuring the lipase activity for short-chain fatty acids, an FCCIIImethod was used. For measuring the lipase activity for long-chain fattyacids, a fat-digesting capacity LMAP method was used. The resultsrevealed that the lipase activity of the cultured supernatant of theCandida cylindracea strain in which the variant lipase (G429M) wasallowed to be expressed was 470 u/mL when the FCIII method was used and155 u/mL when the LMAP method was used (a ratio of short-chain tolong-chain fatty acids=3:1). For comparison, when a measurement was madeof the lipase activity of the cultured supernatant of the parent hoststrain into which the variant lipase gene had been not introduced, theactivity was 267 u/mL when the FCIII method was used and 599 u/mL whenthe LMAP method was used (a ratio of short-chain to long-chain fattyacids=2:5).

As mentioned above, a variant lipase (G429M) specific for short-chainfatty acids was able to be expressed.

(3) Expression of a variant lipase in a filamentous fungus (Aspergillusoryzae) strain A variant lipase (G429M) was expressed using as a host astrain of Aspergillus oryzae that had been made auxotrophic bymutagenesis and by means of using an amylase promoter. A resultingtransformant was cultured under conditions at 30° C. for 76 hours, andlipase activity of the cultured supernatant was measured. For measuringthe lipase activity for short-chain fatty acids, an FCCIII method wasused. For measuring the lipase activity for long-chain fatty acids, afat-digesting capacity LMAP method was used. The results revealed thatthe lipase activity of the cultured supernatant of the Aspergillusoryzae strain in which the variant lipase (G429M) was allowed to beexpressed was 39 u/mL when the FCIII method was used and 0 u/mL when theLMAP method was used.

As mentioned above, a variant lipase (G429M) specific for short-chainfatty acids was able to be expressed.

(4) Expression of a Variant Lipase in a Bacillus subtilis Strain

Into a plasmid pHY300PLK was inserted a variant lipase (G429M) genehaving a pullulanase promoter added thereto. The variant lipase wasexpressed using a Bacillus subtilis strain as a host. Lipase activity ofthe cultured medium of a resulting transformant was measured. Formeasuring the lipase activity for short-chain fatty acids, a Lipase KitS (DS Biopharma Medical) was used. For measuring the lipase activity forlong-chain fatty acids, a fat-digesting capacity LMAP method was used.The results revealed that the lipase activity of the cultured medium was0.3 u/mL (and 0.1 u/mL for a control strain transformed with an emptyvector) when the Lipase Kit S was used and 0 u/mL when the LMAP methodwas used.

As mentioned above, a variant lipase (G429M) specific for short-chainfatty acids was able to be expressed.

INDUSTRIAL APPLICABILITY

The modified lipase according to the present invention exhibitsspecificity for short-chain to medium-chain fatty acids. The modifiedlipase according to the present invention has a great deal of potential,in particular, in the production of dairy products having a cheeseflavor, such as cheeses or cheese products.

The present invention should not be limited in any way to thedescription of the embodiments and examples of the above-describedinvention. The present invention also includes a variety of modifiedembodiments within the scope that one skilled in the art could easilyarrive without departing from the description of the scope of claims.The contents of articles, published patent applications, patentpublications, and others that are expressly provided are incorporated intheir entire content by citation.

The invention claimed is:
 1. A modified lipase consisting of an aminoacid sequence with a substitution made in the amino acid sequence of alipase, wherein the substitution is: (1) a substitution of asparaginefor an amino acid corresponding to the amino acid at position 428 in theamino acid sequence set forth in SEQ ID NO: 1; or (2) a substitution ofphenylalanine, methionine, or isoleucine for an amino acid correspondingto the amino acid at position 429 in the amino acid sequence set forthin SEQ ID NO:1, wherein the amino acid sequence of the lipase is anamino acid sequence that is 90% or more identical to the amino acidsequence of SEQ ID NO. 3 and wherein the substitution is thesubstitution represented in (1), or wherein the amino acid sequence ofthe lipase is an amino acid sequence that is 90% or more identical tothe amino acid sequence of SEQ ID NO. 3 and wherein the substitution isthe substitution represented in (2).
 2. The modified lipase according toclaim 1, wherein the amino acid sequence of the lipase is an amino acidsequence of SEQ ID NO.
 3. 3. A gene encoding the modified lipaseaccording to claim
 1. 4. The gene according to claim 3, comprising thepolynucleotide sequence set forth in SEQ ID No.
 14. 5. A recombinant DNAcomprising the gene according to claim
 3. 6. A microorganism carryingthe recombinant DNA according to claim
 5. 7. The microorganism accordingto claim 6, wherein the host is Escherichia coli, Candida cylindracea,Aspergillus oryzae, Bacillus subtilis, or Pichia pastoris.
 8. An enzymepreparation comprising the modified lipase according to claim
 1. 9. Amethod for improving the flavor of a food product or food raw material,wherein the enzyme according to claim 1 is allowed to act on the foodproduct or food raw material.
 10. A method for producing a food product,wherein the enzyme according to claim 1 is allowed to act on a food rawmaterial or intermediate product.
 11. The method according to claim 9,wherein the food product is a dairy product.
 12. A flavor-improvingagent that is allowed to act on a food product or food raw material,comprising the modified lipase according to claim
 1. 13. A food productor food raw material obtained by treatment with the enzyme according toclaim
 1. 14. The method according to claim 10, wherein the food productis a dairy product.
 15. A flavor-improving agent that is allowed to acton a food product or food raw material, comprising the enzymepreparation according to claim
 8. 16. A modified lipase consisting of anamino acid sequence with a substitution made in the amino acid sequenceof a lipase, wherein the substitution is: (1) a substitution ofasparagine for an amino acid corresponding to the amino acid at position428 in the amino acid sequence set forth in SEQ ID NO: 1; or (2) asubstitution of phenylalanine, methionine, or isoleucine for an aminoacid corresponding to the amino acid at position 429 in the amino acidsequence set forth in SEQ ID NO:1, wherein the amino acid sequence ofthe lipase is an amino acid sequence that is 95% or more identical tothe amino acid sequence of SEQ ID NO: 2 and wherein the substitution isthe substitution represented in (1), or wherein the amino acid sequenceof the lipase is an amino acid sequence that is 95% or more identical tothe amino acid sequence of SEQ ID NO: 2 and wherein the substitution isthe substitution represented in (2).